Method for detecting impurities in propafenone hydrochloride injection
Through high-performance liquid chromatography combined with mobile phase gradient elution method, the detection problems of multiple impurities in propaone hydrochloride injection were solved, and the detection effects of high sensitivity, accuracy and precision were achieved, ensuring the safety and consistency of drug quality.
Patent Information
- Application Number
- CN202510211937.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art lacks effective detection methods to simultaneously detect multiple impurities in propaone hydrochloride injection, making it difficult to ensure the safety and consistency of the quality of the drug.
Using high performance liquid chromatography combined with mobile phase gradient elution method, 12 impurities in propaone hydrochloride injection can be quickly and accurately detected through a special chromatography column and an optimized mobile phase gradient elution program.
High sensitivity, high accuracy and high precision detection of various impurities in propaone hydrochloride injection was achieved, solving the problems of difficulty in baseline separation, poor resolution and uneluting of some impurities, ensuring controllability and consistency of drug quality.
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Abstract
Description
Technical Field
[0001] This application relates to the technical field of chemical detection, and particularly to a method for detecting impurities in propafenone hydrochloride injection. Background Art
[0002] Propafenone hydrochloride injection is an antiarrhythmic drug, mainly used for the prevention of paroxysmal ventricular tachycardia, paroxysmal supraventricular tachycardia, and supraventricular tachycardia, atrial flutter or atrial fibrillation associated with Wolff-Parkinson-White syndrome, and can also be used for the treatment of various premature beats.
[0003] Drug impurities in propafenone hydrochloride injection may have adverse effects on the human body, especially some impurities may be toxic. Through strict impurity detection, these potential harmful substances can be detected and controlled in a timely manner, thus ensuring the safety of patients' medication. Impurity detection is an important part of the quality control system in the pharmaceutical industry; by monitoring the types and contents of impurities, the stability of the production process and the stability of the drug within the validity period can be evaluated, the quality change trend can be detected in a timely manner, and the consistency and controllability of the drug quality can be ensured.
[0004] Currently, there is no mature detection method for the trace detection of multiple impurities in propafenone hydrochloride injection. Therefore, it is very necessary to establish a method for detecting multiple impurities in propafenone hydrochloride injection. Summary of the Invention
[0005] In order to be able to detect multiple impurities in propafenone hydrochloride injection and improve the precision and accuracy of the detection method at the same time, this application provides a method for detecting impurities in propafenone hydrochloride injection.
[0006] This application provides a method for detecting impurities in propafenone hydrochloride injection, adopting the following technical scheme: A method for detecting impurities in propafenone hydrochloride injection specifically includes the following steps: using high performance liquid chromatography to detect the sample to be tested to obtain the content of the impurities; During the high performance liquid chromatography detection process: the chromatographic column uses octadecylsilane bonded silica gel as the filler; the column temperature is 28 - 32 °C, and the flow rate is 0.8 - 1.2 ml / min; gradient elution of the mobile phase is adopted for elution, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a 0.012 - 0.017 mol / L dipotassium hydrogen phosphate solution, and the pH value of the mobile phase A is adjusted to 2.2 - 2.8 with phosphoric acid; the mobile phase B is acetonitrile; The gradient elution method of the mobile phase is: 0 - 14 min, mobile phase A is 65 - 70%, mobile phase B is 30 - 35%; From 14 to 30 min, mobile phase A linearly decreases from 65 - 70% to 30 - 35%, and mobile phase B linearly increases from 30 - 35% to 65 - 70%; from 30 to 40 min, mobile phase A is 30 - 35% and mobile phase B is 65 - 70%. From 40 to 41 min, mobile phase A linearly increases from 30 - 35% to 65 - 70%, and mobile phase B linearly decreases from 65 - 70% to 30 - 35%; from 41 to 50 min, mobile phase A is 65 - 70% and mobile phase B is 30 - 35%.
[0007] Preferably, the impurities include impurity 5 - HMF, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, impurity L. The impurity 5 - HMF is 5 - hydroxymethylfurfural, the impurity A is 1 - (2 - hydroxyphenyl) - 3 - phenyl - 1 - propanone, the impurity B is (2E) - 1 - [2 - [(2RS) - 2 - hydroxy - 3 - propylaminopropoxy]phenyl] - 3 - phenylprop - 2 - en - 1 - one, the impurity C is 1 - [2 - [(2RS) - epoxyethanylmethoxy]phenyl] - 3 - phenyl - 1 - propanone, the impurity D is 1 - [2 - [(2RS) - 2,3 - dihydroxypropoxy]phenyl] - 3 - phenyl - 1 - propanone, the impurity E is 1 - [2 - (2RS) - 3 - chloro - 2 - hydroxypropoxy]phenyl] - 3 - phenyl - 1 - propanone, the impurity F is 1,1′ - [2 - hydroxypropane - 1,3 - diylbis(oxy - 2,1 - phenylene)]bis(3 - phenyl - 1 - propanone), the impurity G is 1,1′ - [propyliminobis[(2 - hydroxypropane - 3,1 - diyl)oxy - 2,1 - phenylene]]bis(3 - phenylpropan - 1 - one), the impurity H is 2 - phenyl - 2,3 - dihydro - 4H - 1 - benzopyran - 4 - one, the impurity J is 1 - (2 - (1 - hydroxy - 3 - propylamino)propan - 2 - oxy)phenyl) - 3 - phenyl - 1 - propanone, the impurity K is 1 - (2 - (3 - amino - 2 - hydroxypropoxy)phenyl) - 3 - phenyl - 1 - propanone, the impurity L is N - (2 - hydroxy - 3 - (2 - (3 - phenylpropanoyl)phenoxy)propyl) - N - propylformamide.
[0008] Preferably, the chromatographic column specification is Thermo Hypersil GOLD C18, 4.6 mm×250 mm, 5 μm.
[0009] Preferably, the column temperature is 29 - 31 °C.
[0010] Preferably, the flow rate is 0.9 - 1.1 ml / min.
[0011] Preferably, the mobile phase gradient elution method is as follows: From 0 to 14 min, mobile phase A is 67% and mobile phase B is 33%; From 14 to 30 min, mobile phase A linearly decreases from 67% to 32%, and mobile phase B linearly increases from 33% to 68%; From 30 to 40 min, mobile phase A is 32% and mobile phase B is 68%; From 40 to 41 min, mobile phase A linearly increases from 32% to 67%, and mobile phase B linearly decreases from 68% to 33%; From 41 to 50 min, mobile phase A is 67% and mobile phase B is 33%.
[0012] Preferably, the pH value of mobile phase A is adjusted to 2.4 - 2.6 with phosphoric acid.
[0013] Preferably, the detection wavelength is 220 nm or 284 nm.
[0014] Preferably, the injection volume is 7 - 13 μl.
[0015] Preferably, during the detection process, the diluent used for the sample is a water - acetonitrile solution with a volume ratio of 62 - 68:32 - 38.
[0016] By using high - performance liquid chromatography and the mobile phase gradient elution method in this application, the content of impurities in the test sample of propafenone hydrochloride injection solution can be quickly obtained. It has a high separation ability for each impurity in the test sample, and then good peak - type parameters can be obtained, so that the detection method provided in this application has the advantages of high sensitivity, high repeatability, high accuracy, and high precision.
[0017] In summary, the technical solution of this application has the following effects: This application provides a method for detecting impurities in propafenone hydrochloride injection. The chromatographic column of this detection method uses octadecylsilane - bonded silica gel as the filler, the column temperature is controlled between 28 - 32 °C, and the mobile phase gradient elution method is adjusted to specific conditions, which can quickly and accurately detect 12 impurities in propafenone hydrochloride injection simultaneously, and the detection has strong specificity, high sensitivity, and good resolution.
[0018] The peaks in the detection chromatogram obtained by the method for detecting impurities in propafenone hydrochloride injection provided in this application have good symmetry, appropriate peak height, centered peak position, and far - apart peak positions of each substance, and the separation effect is obvious. This detection method solves the problems existing in the related technology, such as difficult baseline separation, poor resolution between peaks, and some impurities not being eluted, and then effectively controls the quality of propafenone hydrochloride injection. Brief Description of the Drawings
[0019] Figure 1 It is the chromatogram of the specific mixed reference solution in Example 1 of this application.
[0020] Figure 2 It is the chromatogram of the main component in the linearity and range test in Example 1 of this application.
[0021] Figure 3 It is the chromatogram of 5-HMF in the linearity and range test in Example 1 of this application.
[0022] Figure 4 It is the chromatogram of impurity A in the linearity and range test in Example 1 of this application.
[0023] Figure 5 It is the chromatogram of impurity B in the linearity and range test in Example 1 of this application.
[0024] Figure 6 It is the chromatogram of impurity C in the linearity and range test in Example 1 of this application.
[0025] Figure 7 It is the chromatogram of impurity D in the linearity and range test in Example 1 of this application.
[0026] Figure 8 It is the chromatogram of impurity E in the linearity and range test in Example 1 of this application.
[0027] Figure 9 It is the chromatogram of impurity F in the linearity and range test in Example 1 of this application.
[0028] Figure 10 It is the chromatogram of impurity G in the linearity and range test in Example 1 of this application.
[0029] Figure 11 It is the chromatogram of impurity H in the linearity and range test in Example 1 of this application.
[0030] Figure 12 It is the chromatogram of impurity J in the linearity and range test in Example 1 of this application.
[0031] Figure 13 It is the chromatogram of impurity K in the linearity and range test in Example 1 of this application.
[0032] Figure 14 It is the chromatogram of impurity L in the linearity and range test in Example 1 of this application.
[0033] Figure 15 It is the detection chromatogram in Comparative Example 1 of this application.
[0034] Figure 16 It is the detection chromatogram in Comparative Example 2 of this application.
[0035] Figure 17It is the detection chromatogram in Comparative Example 3 of this application.
[0036] Figure 18 It is the chromatogram of the mixed solution in Comparative Example 3 of this application.
[0037] Figure 19 It is the detection chromatogram in Comparative Example 4 of this application.
[0038] Figure 20 It is the detection chromatogram in Comparative Example 5 of this application.
[0039] Figure 21 It is the detection chromatogram in Comparative Example 6 of this application. Detailed implementation manners
[0040] In this application, the impurity raw materials, their chemical names, chemical structures, and impurity control limit information are specifically shown in Table 1; the raw materials of propafenone hydrochloride injection, impurity reference substances, and their sources used are shown in Table 2; the remaining raw materials, reagents, solvents, etc. can all be obtained through commercial purchase.
[0041] Table 1 Impurity codes and their chemical names, chemical structures, and impurity control limit information Table 2 Propafenone hydrochloride injection, impurity standard solution used in the examples and their sources The following further describes this application in detail in combination with examples and performance detection tests. These examples should not be construed as limiting the scope claimed in this application. Examples
[0042] Example 1 Example 1 provides a method for detecting impurities in propafenone hydrochloride injection.
[0043] In this example, the method for detecting impurities in propafenone hydrochloride injection specifically includes the following steps: Method basis: High performance liquid chromatography.
[0044] Diluent: Water - acetonitrile (65∶35).
[0045] Test solution: Precisely measure 3 ml of this product, place it in a 10 - ml volumetric flask, dilute it to the scale with the diluent, and shake well.
[0046] Reference solution: Weigh accurately appropriate amounts of propafenone hydrochloride reference substance and 5-hydroxymethylfurfural reference substance, dissolve in the diluent and quantitatively dilute to prepare a mixed solution containing about 2 μg of propafenone hydrochloride and 3 μg of 5-hydroxymethylfurfural per 1 ml.
[0047] Sensitivity solution: Accurately measure 1 ml of the test solution, transfer it to a 100-ml volumetric flask, dilute to the mark with the diluent, and shake well; accurately measure 1 ml, transfer it to a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well.
[0048] Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (Thermo Hypersil GOLD C18, 4.6 mm × 250 mm, 5 μm); use 0.015 mol / L dipotassium hydrogen phosphate solution (adjust the pH value to 2.5 with phosphoric acid) as mobile phase A, and acetonitrile as mobile phase B, perform gradient elution according to Table 3 below; the flow rate is 1.0 ml per minute; the column temperature is 30 °C; the detection wavelengths are 220 nm and 284 nm; the injection volume is 10 μl.
[0049] Table 3 Gradient elution method Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 67 33 14 67 33 30 32 68 40 32 68 41 67 33 50 67 33 System suitability requirements: In the chromatogram of the reference solution (220 nm), the elution order is 5-hydroxymethylfurfural and propafenone in sequence, and the number of theoretical plates calculated based on the propafenone peak is not less than 2000; in the chromatogram of the sensitivity solution (220 nm), the signal-to-noise ratio of the main component chromatographic peak height should be greater than 10.
[0050] 1.1 Specificity Examine the specificity of the method. It is required that (1) the blank solution and blank excipients shall not interfere with the inspection of the related substances of this product; (2) in the chromatogram of the mixed reference solution, the resolution between the main component peak and the adjacent peak shall be not less than 1.5; (3) in the chromatogram of the reference solution (220 nm), the elution order is 5-HMF and the main component in sequence, and the number of theoretical plates calculated based on the main component peak is not less than 2000; (4) in the chromatogram of the sensitivity solution (220 nm), the signal-to-noise ratio of the main component chromatographic peak height should be greater than 10.
[0051] Blank solution / diluent: water - acetonitrile (65∶35).
[0052] Blank excipient solution: Accurately measure 3 ml of blank excipients, transfer it to a 10-ml volumetric flask, dilute to the mark with the diluent, and shake well.
[0053] Propafenone hydrochloride stock solution: Weigh accurately about 2 mg of propafenone hydrochloride reference substance, transfer it to a 50-ml volumetric flask, dissolve in the diluent and dilute to the mark, and shake well.
[0054] 5-HMF stock solution: Weigh accurately about 10 mg of 5-HMF reference substance, place it in a 10-ml volumetric flask, dissolve it with the diluent and dilute to the mark, shake well. Accurately measure 3 ml and place it in a 100-ml volumetric flask, dilute to the mark with the diluent and shake well.
[0055] Impurity A stock solution: Weigh accurately about 2 mg of Impurity A reference substance, place it in a 10-ml volumetric flask, dissolve it with acetonitrile and dilute to the mark, shake well.
[0056] Prepare stock solutions of Impurity B, Impurity C, Impurity D, Impurity E, Impurity F, Impurity G, Impurity H, Impurity J, Impurity K and Impurity L in the same way.
[0057] Mixed reference solution: Weigh accurately about 10 mg of propafenone hydrochloride reference substance, place it in a 10-ml volumetric flask, add 1 ml of 5-HMF stock solution, 100 μl each of the stock solutions of Impurity A, Impurity B, Impurity C, Impurity D, Impurity E, Impurity F, Impurity G, Impurity H, Impurity J, Impurity K and Impurity L, dissolve with the diluent and dilute to the mark, shake well.
[0058] Propafenone hydrochloride positioning solution: Take 500 μl of propafenone hydrochloride stock solution and 500 μl of diluent, mix well.
[0059] 5-HMF positioning solution: Take 500 μl of 5-HMF stock solution and 500 μl of diluent, mix well.
[0060] Impurity A positioning solution: Take 100 μl of Impurity A stock solution and 900 μl of diluent, mix well.
[0061] Prepare positioning solutions of Impurity B, Impurity C, Impurity D, Impurity E, Impurity F, Impurity G, Impurity H, Impurity J, Impurity K and Impurity L according to the preparation method of Impurity A positioning solution.
[0062] Reference solution: Accurately measure 1 ml of propafenone hydrochloride stock solution and 2 ml of 5-HMF stock solution, place them in a 20-ml volumetric flask, dilute to the mark with the diluent and shake well.
[0063] Impurity mixed stock solution: Weigh accurately about 2 mg each of the reference substances of Impurity A, Impurity B, Impurity C, Impurity D, Impurity E, Impurity F, Impurity G, Impurity H, Impurity J, Impurity K and Impurity L, place them in a 100-ml volumetric flask, dissolve with an appropriate amount of acetonitrile, dilute to the mark with the diluent and shake well.
[0064] Test solution: Accurately measure 3 ml of this product, place it in a 10-ml volumetric flask, dilute to the mark with the diluent and shake well.
[0065] Test sample spiked solution: Accurately measure 3 ml of this product, place it in a 10-ml volumetric flask, accurately add 1 ml each of the 5-HMF stock solution and the impurity mixed stock solution, dilute to the mark with the diluent, and shake well.
[0066] Sensitivity solution: Accurately measure 1 ml of the test sample solution, place it in a 100-ml volumetric flask, dilute to the mark with the diluent, and shake well; accurately measure 1 ml and place it in a 50-ml volumetric flask, dilute to the mark with the diluent, and shake well.
[0067] Accurately measure the blank solution, blank excipient solution, sensitivity solution, mixed reference solution, reference solution, test sample solution, test sample spiked solution and each localization solution, inject them into the liquid chromatograph, and record the chromatogram. The attached figure is shown in Figure 1 the following. The specificity results are shown in Table 4 Table 4 Method validation for related substances - Specificity Conclusion: The blank solution and blank excipients do not interfere with the inspection of the related substances of this product; in the chromatogram of the mixed reference solution (as shown in Figure 1 the following), the resolution between the main component peak and the adjacent peaks is not less than 1.5 (the minimum value is 2.50); in the chromatogram of the reference solution (220 nm), the elution order is 5-HMF and the main component, and the theoretical plate number of the main component peak is 18184, which is greater than 2000; in the chromatogram of the sensitivity solution (220 nm), the signal-to-noise ratio of the main component chromatographic peak height is 17.0, which is greater than 10; therefore, the specificity of the related substances method of this product is good.
[0068] 1.2 Quantitation limit and detection limit Examine the detection sensitivity of the method, and use the signal-to-noise ratio as the evaluation index. It is required that the corresponding concentration when the signal-to-noise ratio ≥ 10 is used as the quantitation limit, and the corresponding concentration when the signal-to-noise ratio ≥ 3 is used as the detection limit.
[0069] Take appropriate amounts of the propafenone hydrochloride stock solution, 5-HMF stock solution, impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution, impurity E stock solution, impurity F stock solution, impurity G stock solution, impurity H stock solution, impurity J stock solution, impurity K stock solution, and impurity L stock solution under "Specificity" and mix and quantitatively dilute them to prepare the quantitation limit solution and the detection limit solution.
[0070] Accurately measure the quantitation limit solution and the detection limit solution, inject them into the liquid chromatograph, and record the chromatogram.
[0071] The results of the quantitation limit and detection limit are shown in Table 5.
[0072] Table 5 Method validation for related substances - Results of quantitation limit and detection limit Conclusion: The quantitation limits and detection limits of impurities A, B, C, D, E, F, G, H, J, K, L, 5-HMF and the main component all meet the requirements. Therefore, the sensitivity of the related substances method for this product is good.
[0073] 1.3 Linear range Examine the linearity of the contents of each impurity within the range of LOQ to 200% limit. It is required to perform linear regression with the concentration as the abscissa and the peak area as the ordinate, and the correlation coefficient r ≥ 0.998.
[0074] Linear stock solution: Take about 4 mg of each reference substance of propafenone hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K and impurity L, weigh accurately, place them in a 20 ml volumetric flask, dissolve with acetonitrile and dilute to the mark, shake well. Accurately measure 1 ml and place it in a 50 ml volumetric flask, accurately add 10 ml of 5-HMF stock solution, and dilute to the mark with the diluent, shake well.
[0075] Accurately measure the linear stock solution, prepare according to Table 6 below, and dilute with the diluent to make a linear solution.
[0076] Table 6 Preparation plan of linear solution for related substances method validation Accurately measure each linear solution, inject it into the liquid chromatograph, and record the chromatogram. The linearity and range test is as Figures 2 - 14 shown, and the results are shown in Table 7.
[0077] Table 7 Results of linearity and range test for related substances method validation Conclusion: For the related substances of this product, impurities A, B, C, D, E, F, G, H, J, K, L, 5-HMF have a good linear relationship within the range of approximately equivalent to LOQ to 200% limit, meeting the detection requirements.
[0078] 1.4 Accuracy Spiked for accuracy tests of impurities A, B, C, D, E, F, G, H, J, K, and L, each at approximately 0.1%, 0.2%, 0.3% (i.e., 50%, 100%, 150% limits), and for 5-HMF at approximately 0.01%, 0.02%, 0.03% (i.e., 50%, 100%, 150% limits). When each impurity is at approximately 50%, 100%, 150% limits, the average recovery rate should be between 80% and 120%, and RSD (n = 9) ≤ 10%.
[0079] Blank solution / diluent: water - acetonitrile (65∶35).
[0080] Propafenone hydrochloride stock solution: Take about 2 mg of propafenone hydrochloride reference substance, weigh accurately, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with diluent, and shake well.
[0081] 5-HMF stock solution: Take about 10 mg of 5-HMF reference substance, weigh accurately, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, accurately measure 3 ml, place it in a 100 ml volumetric flask, and dilute to the mark with diluent, and shake well.
[0082] Reference solution: Accurately measure 1 ml of propafenone hydrochloride stock solution and 2 ml of 5-HMF stock solution, place them in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.
[0083] Impurity mixed stock solution: Take 2 mg each of reference substances of impurities A, B, C, D, E, F, G, H, J, K, and L, weigh accurately, place them in a 100 ml volumetric flask, add an appropriate amount of acetonitrile to dissolve, and dilute to the mark with diluent, and shake well.
[0084] Impurity reference solution: Accurately measure 1 ml of impurity mixed stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.
[0085] Test solution: Accurately measure 3 ml of this product, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.
[0086] 50% concentration - test solution spiked solution: Accurately measure 3 ml of this product, place it in a 10 ml volumetric flask, accurately add 0.5 ml each of 5-HMF stock solution and impurity mixed stock solution, dilute to the mark with diluent, and shake well. Prepare 3 parallel portions.
[0087] 100% concentration - test solution spiked solution: Accurately measure 3 ml of this product, place it in a 10 ml volumetric flask, accurately add 1 ml each of 5-HMF stock solution and impurity mixed stock solution, dilute to the mark with diluent, and shake well. Prepare 3 parallel portions.
[0088] 150% Concentration - Spiked Test Solution: Accurately measure 3 ml of this product and transfer it to a 10-ml volumetric flask. Accurately add 1.5 ml each of the 5-HMF stock solution and the impurity mixed stock solution, dilute to the mark with the diluent, and mix well. Prepare 3 parallel portions.
[0089] Accurately measure the blank solution, reference solution, impurity reference solution, test solution, and spiked test solutions at each concentration level, inject them into the liquid chromatograph, and record the chromatograms.
[0090] Accuracy - The results of 5-HMF, Impurity A, Impurity B, Impurity C, Impurity D, Impurity E, Impurity F, Impurity G, Impurity H, Impurity J, Impurity K, and Impurity L are shown in Table 8 in sequence.
[0091] Table 8 Validation of Related Substances Method - Accuracy Conclusion: By using the external standard method, the average recoveries of Impurity A, Impurity B, Impurity C, Impurity D, Impurity E, Impurity F, Impurity G, Impurity H, Impurity J, Impurity K, and Impurity L at approximately 50%, 100%, and 150% limits are all within the range of 80% - 120%. The average recovery of 5-HMF calculated by the external standard method is 101.0%, within the range of 80% - 120%, and the RSDs are all less than 10% (the maximum value is 3.15%). Therefore, the accuracy of the related substances method for this product is good.
[0092] 1.5 Robustness Investigate the influence of minor changes in column temperature, mobile phase pH value, and chromatographic column in the chromatographic conditions on the detection results of the related substances of this product. It is required that the blank solution and blank excipients should not interfere with the inspection of the related substances of this product under each chromatographic condition; in the chromatogram of the reference solution (220 nm), the elution order is 5-HMF and the main component in sequence, and the theoretical plate number calculated based on the main component peak is not less than 2000; in the chromatogram of the sensitivity solution (220 nm), the signal-to-noise ratio of the main component chromatographic peak should be greater than 10; the RSD of the detected amounts of each impurity in the spiked test solution of the test sample ≤ 10%. The conditions for the robustness test are shown in Table 9.
[0093] Table 9 Validation of Related Substances Method - Conditions for Robustness Test Blank Solution / Diluent: Water - Acetonitrile (65∶35).
[0094] The preparation methods of the blank excipient solution, propafenone hydrochloride stock solution, 5-HMF stock solution, reference solution, impurity mixed stock solution, spiked test solution of the test sample, and sensitivity solution are the same as those under "Specificity".
[0095] Accurately measure the blank solution, blank excipient solution, sensitivity solution, reference solution, and spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. The durability - system suitability test is shown in Table 10; the durability - spiked test solution is shown in Table 11; the durability - spiked test solution - 5 - HMF is shown in Table 12.
[0096] Table 10 Validation of Related Substances Methodology - Durability - System Suitability Test Table 11 Validation of Related Substances Methodology - Durability - Spiked Test Solution (%) Note: No other single impurity was detected.
[0097] Table 12 Validation of Related Substances Methodology - Durability - Spiked Test Solution - 5 - HMF Conclusion: When the column temperature is (28°C - 32°C), the pH value of the mobile phase is (2.2 - 2.8), and different chromatographic columns are used, the blank solution and blank excipient do not interfere with the inspection of related substances of this product; in the chromatogram of the reference solution, the theoretical plate number of the propafenone peak is greater than 2000 (the minimum value is 17817); in the chromatogram of the sensitivity solution (220 nm), the signal - to - noise ratio of the main component chromatographic peak height is greater than 10 (the minimum value is 15.8); the RSD of the detected amounts of each impurity in the spiked test solution is less than 10% (the maximum value is 6.07%), and the detection results of related substances are basically the same. Therefore, slightly changing the column temperature, pH value of the mobile phase, and chromatographic column in the chromatographic conditions does not affect the detection results of related substances of this product, and the method has good durability.
[0098] Example 2 Example 2 provides a method for detecting impurities in propafenone hydrochloride injection.
[0099] The difference between this example and Example 1 is that the mobile - phase gradient elution method is as follows: from 0 - 14 min, mobile phase A is 65% and mobile phase B is 35%; from 14 - 30 min, mobile phase A linearly decreases from 65% to 30%, and mobile phase B linearly increases from 35% to 70%; from 30 - 40 min, mobile phase A is 30% and mobile phase B is 70%; from 40 - 41 min, mobile phase A linearly increases from 30% to 65%, and mobile phase B linearly decreases from 70% to 35%; from 41 - 50 min, mobile phase A is 65% and mobile phase B is 35%; the remaining parameters in this example are the same as those in Example 1.
[0100] Conclusion: Under these chromatographic conditions, the resolution between impurity peaks and between the main component and adjacent impurity peaks basically meets the requirements, and the method is feasible. In the specificity test, the blank solution and blank excipients do not interfere with the determination of related substances in this product; in the chromatogram of the mixed reference solution, the resolution between the main component peak and adjacent peaks is not less than 1.5; in the chromatogram of the reference solution (220 nm), the elution order is 5-HMF and the main component, and the theoretical plate number of the main component peak is greater than 2000; in the chromatogram of the sensitivity solution (220 nm), the signal-to-noise ratio of the main component chromatographic peak height is greater than 10; therefore, the specificity of the related substances method for this product is good.
[0101] Example 3 Example 3 provides a method for detecting impurities in propafenone hydrochloride injection.
[0102] The difference between this example and Example 1 is that the mobile phase gradient elution method is as follows: from 0 to 14 min, mobile phase A is 70% and mobile phase B is 30%; from 14 to 30 min, mobile phase A linearly decreases from 70% to 35% and mobile phase B linearly increases from 30% to 65%; from 30 to 40 min, mobile phase A is 35% and mobile phase B is 65%; from 40 to 41 min, mobile phase A linearly increases from 35% to 70% and mobile phase B linearly decreases from 65% to 30%; from 41 to 50 min, mobile phase A is 70% and mobile phase B is 30%; the remaining parameters in this example are the same as those in Example 1.
[0103] Conclusion: Under these chromatographic conditions, the resolution between impurity peaks and between the main component and adjacent impurity peaks basically meets the requirements, and the method is feasible. In the specificity test, the blank solution and blank excipients do not interfere with the determination of related substances in this product; in the chromatogram of the mixed reference solution, the resolution between the main component peak and adjacent peaks is not less than 1.5; in the chromatogram of the reference solution (220 nm), the elution order is 5-HMF and the main component, and the theoretical plate number of the main component peak is greater than 2000; in the chromatogram of the sensitivity solution (220 nm), the signal-to-noise ratio of the main component chromatographic peak height is greater than 10; therefore, the specificity of the related substances method for this product is good.
[0104] Comparative Example Comparative Example 1 Comparative Example 1 provides a method for detecting impurities in propafenone hydrochloride injection.
[0105] This example was detected according to ChP2020 for propafenone hydrochloride injection, and the specific method steps are as follows: High Performance Liquid Chromatography: Chromatographic column: octadecylsilyl silica gel (Agilent ZORBAX Eclipse XDB-C8, 4.6 mm × 250 mm, 5 μm). Isocratic elution; Mobile phase: 0.0015 mol / L dipotassium hydrogen phosphate (adjusted to pH 2.5 with phosphoric acid)-acetonitrile (65:35); Flow rate: 1.0 ml / min; Column temperature: 30 °C; Detection wavelength: 220 nm; Injection volume: 10 μl.
[0106] System suitability requirements: The number of theoretical plates calculated for the propafenone peak is not less than 2000.
[0107] The detected chromatographic results are as Figure 15 shown.
[0108] Conclusion: The number of theoretical plates of the propafenone peak is 305, less than 2000, not meeting the system suitability requirements in ChP2020, so this chromatographic column is not applicable.
[0109] Comparative Example 2 Comparative Example 2 provides a method for detecting impurities in propafenone hydrochloride injection.
[0110] In this example, the detection was carried out according to ChP2020 for propafenone hydrochloride injection. The specific method steps are as follows: High Performance Liquid Chromatography: Octadecylsilyl silica gel (YMC Pack Pro C8, 4.6 mm × 150 mm, 5 μm). Isocratic elution; Mobile phase: 0.0015 mol / L dipotassium hydrogen phosphate (adjusted to pH 2.5 with phosphoric acid)-acetonitrile (65:35); Flow rate: 1.0 ml / min; Column temperature: 30 °C; Detection wavelength: 220 nm; Injection volume: 10 μl.
[0111] System suitability requirements: The number of theoretical plates calculated for the propafenone peak is not less than 2000.
[0112] The detected chromatogram is as Figure 16 shown.
[0113] Conclusion: It can be seen from the chromatogram that the number of theoretical plates of the propafenone peak is 896, less than 2000, not meeting the system suitability requirements in ChP2020, and the retention time of the main peak is relatively short, which is not conducive to the separation and detection of impurities. Therefore, this chromatographic column is not applicable.
[0114] Comparative Example 3 Comparative Example 3 provides a method for detecting impurities in propafenone hydrochloride injection.
[0115] In this example, the detection was carried out according to ChP2020 for propafenone hydrochloride injection. The specific method steps are as follows: High performance liquid chromatography: Octadecylsilyl silica gel (COSMOSIL 5C8-MS, 4.6 mm × 250 mm, 5 μm). Isocratic elution; Mobile phase: 0.0015 mol / L dipotassium hydrogen phosphate (adjusted to pH 2.5 with phosphoric acid) - acetonitrile (65:35); Flow rate: 1.0 ml / min; Column temperature: 30 °C; Detection wavelength: 220 nm; Injection volume: 10 μl.
[0116] System suitability requirements: The number of theoretical plates calculated by propafenone peak should be not less than 2000.
[0117] The detection chromatogram is as Figure 17 shown.
[0118] Conclusion: The number of theoretical plates of propafenone peak is 1290, which is relatively high. Considering injecting the mixed solution under this chromatographic condition to investigate the specificity.
[0119] The chromatogram of the mixed solution is as Figure 18 shown.
[0120] Conclusion: Under this chromatographic condition, the running time is long, the impurity peak shape is poor, and the sensitivity is low, which is not conducive to detection. Therefore, this method is not applicable.
[0121] Comparative Example 4 Comparative Example 4 provides a method for detecting impurities in propafenone hydrochloride injection.
[0122] In this example, the detection was carried out according to ChP2020 of propafenone hydrochloride injection. The specific method steps are as follows: In high performance liquid chromatography, the chromatographic column is: Octadecylsilyl silica gel (Welch Ultimate XB-C8, 4.6 mm × 250 mm, 5 μm). The other conditions are the same as those in Example 1.
[0123] The detection chromatogram is as Figure 19 shown.
[0124] Conclusion: Under this chromatographic condition, the retention times of impurity G and impurity H are basically the same, and they elute together. Therefore, this chromatographic column is not applicable.
[0125] Comparative Example 5 Comparative Example 5 provides a method for detecting impurities in propafenone hydrochloride injection.
[0126] In this example, the detection was carried out according to ChP2020 of propafenone hydrochloride injection. The specific method steps are as follows: In high performance liquid chromatography, the chromatographic column is: Octadecylsilyl silica gel (Phenomenex LC C8, 4.6 mm × 250 mm, 5 μm). The other conditions are the same as those in Example 1.
[0127] The detection chromatogram is as follows Figure 20 as shown
[0128] Conclusion: Under these chromatographic conditions, the resolution between the main peak and the adjacent impurity peaks is small, so these conditions are not applicable
[0129] Comparative Example 6 Comparative Example 6 provides a method for detecting impurities in propafenone hydrochloride injection
[0130] In this example, the detection was carried out according to ChP2020 for propafenone hydrochloride injection. The specific method steps were as follows: High performance liquid chromatography: octadecylsilane chemically bonded silica gel (Phenomenex Luna C18, 4.6 mm×250 mm, 5 μm); Considering the detection sensitivity of each impurity and referring to ChP2020, the injection volume was adjusted to 10 μl, and the gradient elution program was adjusted simultaneously. The elution gradient method is shown in Table 13. The other conditions were the same as those in Example 1
[0131] Table 13 Elution gradient method in Comparative Example 6 The detection chromatogram is as follows Figure 21 as shown
[0132] Conclusion: Under these chromatographic conditions, the resolution between the main peak and the adjacent impurity peaks meets the requirements, but the resolution between the impurities is slightly poor, so these conditions are not applicable
[0133] Comparative Example 7 Comparative Example 7 provides a method for detecting impurities in propafenone hydrochloride injection
[0134] The difference between this comparative example and Example 1 is that the column temperature is 40 °C
[0135] Conclusion: Under these chromatographic conditions, impurity E coincides with impurity C, so these conditions are not applicable
[0136] Comparative Example 8 Comparative Example 8 provides a method for detecting impurities in propafenone hydrochloride injection
[0137] The difference between this comparative example and Example 1 is that the gradient elution program is as follows: 0 - 14 min, mobile phase A is 60%, mobile phase B is 40%; 14 - 30 min, mobile phase A linearly decreases from 60% to 30%, mobile phase B linearly increases from 40% to 70%; 30 - 40 min, mobile phase A is 30%, mobile phase B is 70%; 40 - 41 min, mobile phase A linearly increases from 30% to 60%, mobile phase B linearly decreases from 70% to 40%; From 41 to 50 min, mobile phase A is 60% and mobile phase B is 40%.
[0138] Conclusion: Under this chromatographic condition, the peak emergence time of 5-HMF is relatively early, which is not conducive to the detection of impurities. Therefore, this condition is not applicable.
[0139] Comparative Example 10 Comparative Example 10 provides a method for detecting impurities in propafenone hydrochloride injection.
[0140] The difference between this comparative example and Example 1 lies in that the gradient elution program is from 0 to 10 min, mobile phase A is 67% and mobile phase B is 33%; From 10 to 30 min, mobile phase A linearly decreases from 67% to 32%, and mobile phase B linearly increases from 33% to 68%; From 30 to 40 min, mobile phase A is 32% and mobile phase B is 68%; From 40 to 50 min, mobile phase A linearly increases from 32% to 67%, and mobile phase B linearly decreases from 68% to 33%; From 50 to 60 min, mobile phase A is 67% and mobile phase B is 33%.
[0141] Conclusion: Under this chromatographic condition, impurity E coincides with impurity C. Therefore, this condition is not applicable.
[0142] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.
Claims
1. A method for detecting impurities in propafenone hydrochloride injection, characterized in that: The specific steps include: Detecting the sample by high performance liquid chromatography to obtain the content of the impurity; During the high performance liquid chromatography detection process: the chromatographic column uses octadecylsilane bonded silica gel as a filler; the column temperature is 28-32°C, and the flow rate is 0.8-1.2 ml / min; the mobile phase gradient elution method is used for elution, and the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is 0.012-0.017 mol / L dipotassium hydrogen phosphate solution, and the mobile phase A is adjusted to a pH value of 2.2-2.8 with phosphoric acid; the mobile phase B is acetonitrile; The mobile phase gradient elution method is: 0-14min, mobile phase A is 65-70%, mobile phase B is 30-35%; From 14 to 30 min, mobile phase A was linearly reduced from 65-70% to 30-35%, and mobile phase B was linearly increased from 30-35% to 65-70%; 30-40min, mobile phase A is 30-35%, mobile phase B is 65-70%; At 40-41 min, mobile phase A increased linearly from 30-35% to 65-70%, and mobile phase B decreased linearly from 65-70% to 30-35%; 41-50min, mobile phase A is 65-70%, mobile phase B is 30-35%.
2. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: The impurities include impurity 5-HMF, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity J, impurity K, and impurity L; The impurity 5-HMF is 5-hydroxymethylfurfural, the impurity A is 1-(2-hydroxyphenyl)-3-phenyl-1-propanone, the impurity B is (2E)-1-[2-[(2RS)-2-hydroxy-3-propylaminopropoxy]phenyl]-3-phenylprop-2-ene-1-one, the impurity C is 1-[2-[(2RS)-oxiranyl]methoxy]phenyl]-3-phenyl-1-propanone, the impurity D is 1-[2-[(2RS)-2,3-dihydroxypropoxy]phenyl]-3-phenyl-1-propanone, the impurity E is 1-[2-(2RS)-3-chloro-2-hydroxypropoxy]phenyl]-3-phenyl-1-propanone, the impurity F is 1,1′-[2-hydroxypropane-1,3 -diylbis(oxy-2,1-phenylene)]bis(3-phenyl-1-propanone), the impurity G is 1,1′-[propyliminobis[(2-hydroxypropane-3,1-diyl)oxy-2,1-phenylene]]bis(3-phenylpropane-1-one), the impurity H is 2-phenyl-2,3-dihydro-4H-1-benzopyran-4-one, the impurity J is 1-(2-(1-hydroxy-3-propylamino)propan-2-oxy)phenyl)-3-phenyl-1-propanone, the impurity K is 1-(2-(3-amino-2-hydroxypropoxy)phenyl)-3-phenyl-1-propanone, and the impurity L is N-(2-hydroxy-3-(2-(3-phenylpropionyl)phenoxy)propyl)-N-propylformamide.
3. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: The specifications of the chromatographic column are Thermo Hypersil GOLD C18, 4.6 mm×250 mm, 5 μm.
4. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: The column temperature was 29-31°C.
5. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: The flow rate is 0.9-1.1 ml / min.
6. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: The mobile phase gradient elution method is: 0-14min, mobile phase A was 67%, mobile phase B was 33%; From 14 to 30 min, mobile phase A decreased linearly from 67% to 32%, and mobile phase B increased linearly from 33% to 68%; 30-40 min, mobile phase A is 32%, mobile phase B is 68%; At 40-41 min, mobile phase A increased linearly from 32% to 67%, and mobile phase B decreased linearly from 68% to 33%; 41-50min, mobile phase A was 67% and mobile phase B was 33%.
7. The method for detecting impurities in propafenone hydrochloride injection according to any one of claim 1, characterized in that: The pH value of the mobile phase A was adjusted to 2.4-2.6 with phosphoric acid.
8. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: The detection wavelength is 220nm or 284nm.
9. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: The injection volume was 7-13 μl.
10. The method for detecting impurities in propafenone hydrochloride injection according to claim 1, characterized in that: During the detection process, the diluent used for the sample was a water-acetonitrile solution with a volume ratio of 62-68:32-38.
Citation Information
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